Controlling vital natural killer (NK) cell functions in immunity and reproduction are variable receptors that recognize polymorphic determinants of major histocompatibility complex (MHC) class I molecules. These ligands and receptors vary greatly between mammalian species, notably between humans and mice, necessitating in-depth analysis of the human system for understanding its basic biology as well as for the development of clinically applicable NK-cell based diagnostics and therapeutics. The human killer-cell immunoglobulin-like receptors (KIR) recognize epitopes of the human MHC class I molecules HLA-A, -B and -C, but it is HLA-C that has evolved to dominate KIR-mediated regulation of human NK cells and it will therefore be the focus for this program of research. Originally described as a bipartite system of two mutually exclusive ligand-receptor pairs, research accomplished over the last six years, with the development and application of more accurate and sensitive methods, points to a greater complexity. KIR polymorphism, HLA-C polymorphism and sequence variability in the peptide bound to MHC-C, are all seen to influence the interactions between KIR and HLA-C, their functional effects, and associations with a wide range of diseases. Aim 1 will determine how worldwide variation in the five KIR that recognize HLA-C (inhibitory 2DL1, 2DL2 and 2DL3, and activating KIR2DS1 and 2DS4) modulates their strength and specificity for the C1 and C2 epitopes of HLA-C, as well as reactivity with HLA-A and -B. Aim 3 will employ innovative methodology to define how sequence variation within the many thousands of peptides that can be bound by HLA-C affects the interactions with KIR. Such analysis will assess the relative frequency of permissive and non-permissive bound peptides and determine if certain activating human KIR, notably 2DS2, 2DS3 and 2DS5, which appear unable to bind to HLA-C are in fact highly peptide-specific. Aim 2 will apply a new form of cytometry, which has much greater resolution and analytical capability than conventional flow cytometry, to examine the heterogeneity of human NK cells and their varied expression of KIR and other receptors. By studying these NK-cell receptor repertoires in cohorts of human blood donors, variation within the human population will be defined and correlated with both KIR and HLA class I genotypes, and their combination. A particular focus for study is to compare the influences on NK- cell receptor repertoire of the KIR A and B haplotypes that have been shown to differentially associate in numerous studies of human disease. These studies of the molecular and cellular variability of NK cells and their receptors should provide insights to the variabilityof the human NK response to infection, cancer, and the allogeneic cells encountered naturally in pregnancy and clinically in tissue and organ transplantation.